Non-A, non-B hepatitis (NANBH) remains the most common form of post-transfusion hepatitis, imposing a strong need for sensitive and specific diagnostic screening methods to identify potential blood donors and other persons who may be carriers of the disease. Thus, accurate screening methods are needed to permit removal of contaminated blood and blood products from the blood supply with a high degree confidence.
The etiological agent of NANBH, HCV, has been cloned and identified by several groups [Houghton et al., EP 0318216, published 5/1989; Okamoto et al. (1990) Jpn. J. Exp. Med. 60:167; Houghton et al., EP 0388232, published 9/1990; and Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87:9524; Arima et al. (1989a) Gastroenterologia Japonica 24:540; Reyes et al. (1990) Science 247:1335; Arima et al. (1989b) Gastroenterologia Japonica 24:545; Maeno et al. (1990) Nucleic Acids Res. 18:2685]. The HCV genome is about 10 kilobases (kb) in length and encodes a single polyprotein which is processed into structural and non-structural proteins. From the N terminus, the polyprotein includes the capsid and envelope proteins of the structural region and the NS-1 to NS-5 proteins of the non-structural region.
While some of the antigenic regions of HCV have been identified, peptides and recombinant proteins from these regions exhibit a variable degree of sensitivity and selectivity in detection and diagnosis of NANBH carriers. Antigenic regions have been reported in the core, or capsid, protein [Hosein et al. (1991) Proc. Natl. Acad. Sci. USA 88:3647; UBI HCV EIA Product Insert (1990); Okamoto et al. (1990) Jap. J. Exp. Med. 60:223; U.S. Pat. No. 5,106,726; Takahashi et al. (1992) J. Gen. Virol. 73:667; Kotwal et al. (1992) Proc. Natl. Acad. Sci. USA 89:4486]; in the envelope, NS-1, NS-2 and NS-3 proteins [Wang et al., EP 0468527, published Jan. 29, 1992]; NS-4 protein [Houghton (1989); Kuo et al. (1989) Science 244:362; U.S. Pat. No. 5,106,726] and NS-5 protein [Maeno et al. (1990) Nucleic Acids Res. 18:2685; Wang (1992)].
In addition to HCV-derived antigens, there exist other NANBH-associated antigens that appear to be encoded by a host cellular sequence. One such antigen, known as the GOR epitope, is reactive with sera from individuals who are PCR positive for HCV [Mishiro et al. (1990) Lancet 336:1400].
Serological validation has been used to map epitopes within certain HCV antigenic regions as described in Wang (1992) and U.S. Pat. No. 5,106,726, each of which is incorporated herein by reference. These mapping studies employed synthetic peptides to screen well-characterized NANBH serum panels and permitted identification of strong HCV antigens. Further refinement of the epitope analysis using serological validation techniques has led to the discovery that small clusters of amino acid residues contained within longer branched peptides or fusions of peptides containing one or more epitopes from separate regions of the HCV genome provide a superior and more sensitive assay for diagnosis and detection of NANBH carriers as well as for HCV infection. Hence, the present invention permits earlier detection of NANBH seroconversion and shows improved specificity, for example, fewer false positive serum samples are detected.